PROJECT SUMMARY Identifying the neural circuit mechanisms underlying both homeostatic and uncontrolled motivation are crucial for developing more targeted and effective therapeutic treatments for neuropsychiatric disorders like major depression or bipolar disorder. Within mesolimbic brain reward circuitry, the ventral tegmental area (VTA) is a major regulator of both reward and aversion. While generally reduced to its dopamine projection neurons, the VTA also contains neurons that co-release inhibitory GABA and excitatory glutamate and, similar to dopamine projections, can modulate motivation. However, the functional role of this GABA/glutamate co- release is still not well understood, and major questions regarding their role in positive vs. negative reinforcement remain. In particular, why would neurons simultaneously send an inhibitory and excitatory signal? This set of proposed experiments will systematically test the hypothesis that VTA GABA/glutamate co- release serves as a mechanism to balance postsynaptic activity and motivation in a homeostatic manner. Using optogenetics to drive VTA GABA/glutamate co-release in downstream structures that regulate mood and motivation, I will apply gene-editing tools (CRISPR/Cas9) to test the selective contribution of GABA vs. glutamate release in behavioral reinforcement (Aim 1). I will then apply chemogenetic approaches and chronic behavioral manipulations that alter postsynaptic activity to test the net in vivo effect of optogenetically driving VTA GABA/glutamate co-release using in vivo calcium imaging (Aim 2). These experiments will reveal basic mechanisms of neurotransmitter co-release and uncover their role within mesolimbic circuitry in balancing motivation and preventing prolonged excitation/inhibition of efferent structures associated with extreme motivational states. The capability of GABA/glutamate co-release to normalize activity can provide a novel target for treating disorders of imbalanced motivation such as bipolar disorder or depression. A strong interdisciplinary team of faculty mentors and experts will provide the scientific training required for these proposed experiments (gene-editing and fiber photometry) as well as professional development training to facilitate a successful transition to the R00 phase of this proposal. During the R00 phase, I will unify my prior expertise with newly acquired skills to determine the physiological and behavioral role of a newly-described VTA GABA/glutamate projection to amygdala. Using in vivo calcium imaging, I will first monitor activity of VTA GABA/glutamate terminals in amygdala during an effort-based instrumental sucrose task, and will then identify the net effect of optogenetically stimulating VTA GABA/glutamate terminals on cell-defined amygdala populations (Aim 3). These results will significantly expand our understanding of the role of VTA co-release in complex motivated behavior relevant to models of bipolar disorder and depression, and wi...